User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/09/30: Difference between revisions

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[[Image:CalciumCC.png]]<br.>
**Try measuring 50 μM and 50 mM of other salts to check for interferences (Mg<sup>2+</sup> should, Na<sup>+</sup> should not)
**Try measuring 50 μM and 50 mM of other salts to check for interferences (Mg<sup>2+</sup> should, Na<sup>+</sup> should not)
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''CaCl<sub>2</sub> concentration'''
| align="center" style="background:#f0f0f0;"|'''Cu(II)Cl<sub>2</sub> concentration'''
| align="center" style="background:#f0f0f0;"|'''KCl concentration'''
| align="center" style="background:#f0f0f0;"|'''mV measurement (mV)'''
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| 50mM || 0||50mM||86.4
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| 50mM || 50mM||0||80.5
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'''Note''': There may be minimal interference by Cl<sup>-</sup>
**Be sure to measure HPLC water, DI water, tap water, and your lysozyme stock solution.
**Be sure to measure HPLC water, DI water, tap water, and your lysozyme stock solution.
{| {{table}}
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[[Image:ClCC.png]]
**Try measuring 50 μM and 50 mM of other salts to check for interferences & ion-pairing
**Try measuring 50 μM and 50 mM of other salts to check for interferences & ion-pairing
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''NaCl concentration'''
| align="center" style="background:#f0f0f0;"|'''NaCl concentration'''
| align="center" style="background:#f0f0f0;"|'''Cu(II)Cl<sub>2</sub> concentration'''
| align="center" style="background:#f0f0f0;"|'''NaSO<sub>4</sub>'''
| align="center" style="background:#f0f0f0;"|'''KCl concentration'''
| align="center" style="background:#f0f0f0;"|'''Na<sub>2</sub>HPO<sub>4</sub><sup>2-</sup>'''
| align="center" style="background:#f0f0f0;"|'''mV measurement (mV)'''
| align="center" style="background:#f0f0f0;"|'''mV measurement (mV)'''
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| 50mM || 50 mM||0||-7.7
| 50mM || 50 mM||0||-27.0
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| 50μM || 50 μM||0||111.1
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| 50mM || 0||50mM||-43.3
| 50mM || 0||50mM||-30.8
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| 50μM || 0||50μM||115.6
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**Be sure to measure HPLC water, DI water, tap water, and your lysozyme stock solution.
**Be sure to measure HPLC water, DI water, tap water, and your lysozyme stock solution.
{| {{table}}
{| {{table}}

Revision as of 12:28, 1 October 2014

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Tasks for September 30

  • To run dyalisis
  • To prepare calibration curves Ca2+ and Cl-

Making up calibration curves for Ca2+ and Cl-

Procedure followed as described by Dr. Fox

  • Ca2+ calibration curve
    • Prepare 25 mL 50 mM CaCl2 using HPLC water
      • Added 0.13876g CaCl2 to a 25 mL volumetric flask and made up to the mark with HPLC
    • Using serial dilutions, prepare 25 mL each of 5 mM, 500 μM, 50 μM, & 5 μM CaCl2
    • Measure Ca2+ using ISE
CaCl2 concentration mV measurement (mV)
5μM -44.6
50μM 8.2
500μM 39.5
5mM 63.5
50mM 86.6

<br.>

    • Try measuring 50 μM and 50 mM of other salts to check for interferences (Mg2+ should, Na+ should not)
CaCl2 concentration Cu(II)Cl2 concentration KCl concentration mV measurement (mV)
50mM 0 50mM 86.4
50mM 50mM 0 80.5

Note: There may be minimal interference by Cl-

    • Be sure to measure HPLC water, DI water, tap water, and your lysozyme stock solution.
Substance mV measurement (mV)
Tap water 51.3
HPLC water -23.0
DI water -51.5
Lysozyme 0.5g/L -19.1
  • Cl- calibration curve
    • Prepare 25 mL 50 mM NaCl using HPLC water
      • Added 0.07091 g NaCl to a 25 mL volumetric flask and made up to the mark with HPLC
    • Using serial dilutions, prepare 25 mL each of 5 mM, 500 μM, 50 μM, & 5 μM NaCl
    • Measure Cl- using ISE
NaCl concentration mV measurement (mV)
5μM 146.5
50μM 129.6
500μM 81.3
5mM 27.5
50mM -23.9

    • Try measuring 50 μM and 50 mM of other salts to check for interferences & ion-pairing
NaCl concentration NaSO4 Na2HPO42- mV measurement (mV)
50mM 50 mM 0 -27.0
50mM 0 50mM -30.8
    • Be sure to measure HPLC water, DI water, tap water, and your lysozyme stock solution.
Substance mV measurement (mV)
Tap water 65.5
HPLC water 163.5
DI water 158.8
Lysozyme 0.5g/L 89.4

Dyalisis

  1. Dialysis
    • Prepare 10 mL 0.5 g/L Lysozyme
      • Added 0.0050 g to 10mL volumetric flask and made up to mark with HPLC
    • Prepare 2 mL 50 mM CaCl2 (if you read ahead, you can use your CaCl2 calibration stock)
    • Prepare 2 mL 500 μM CaCl2 (if you read ahead, you can use a CaCl2 calibration dilution)
    • Prepare 2 mL 50 mM NaCl (if you read ahead, you can use your NaCl calibration stock)
    • Prepare 1 mL 0.25 mM HCl using your 2.5 mM stored stock
    • Cut a piece of 3500 MWCO flat dialysis tubing, wet it, and insert it in the 5-well dialysis chamber
    • Clamp the dialysis chamber shut
    • Fill one side of each well with 1 mL of your 0.5 g/L Lysozyme solution
      • Transfer 1 mL to a plastic test tube using a pippetter, then
      • Carefully fill each well with with a glass Pasteur pipette
      • You may need to tip the chamber on an angle to reduce water surface tension
    • Fill the other side of the well with one of the following solutions
      • HPLC water, 0.25 mM HCl, 50 mM CaCl2, 500 μM CaCl2, or 50 mM NaCl
      • Each well has a different solution, which will be tested tomorrow
    • Insert top screws to prevent leaking/evaporation
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